Numerous assays have been described which make use of the specific binding properties of certain molecules to detect the presence of an analyte of interest in a sample. Typically such assays involve the specific binding between immunoglobulins (such as antibodies or functional binding fragments thereof) and haptens or antigens to which the immunoglobulins bind. Examples of such assays include enzyme-linked immunosorbent assays (ELISAs) and radio-immunoassay (RIA).
Conventionally, in order to detect binding between the analyte of interest and a binding partner having specific binding affinity therefor, it is necessary for the binding partner to be labelled. Known labels include enzymes, radio-labels, fluorescent or chemiluminescent labels, electroactive labels (such as redox labels) and coloured particles (e.g. latex beads).
A refinement of assays of the general nature outlined above relates to "displacement" assays. In such assays, the presence of an analyte of interest in a sample causes the displacement either of a labelled binding partner or a labelled ligand from a pre-existing binding partner/ligand complex. Generally speaking the amount of displaced labelled substance will be proportional to the concentration of the analyte of interest in the sample. Alternatively, one may employ "competition" assays, in which there is competition between the analyte of interest and a labelled competitor (such as labelled analyte or analogue) for binding to available binding sites.
Several assay methods relying on competition and/or displacement are described in the prior art. For example, EP 0,324,540 discloses assays designed to measure the amount of free ligand (rather than complexed ligand, which complexed ligand is typically protein-bound) in biological samples such as plasma or serum. The assay method requires the use of a "signal reagent", which is a labelled monoclonal antibody. The monoclonal binds to free ligand, which is in competition with a ligand analogue (which analogue does not bind to the natural ligand complexing proteins present in the sample). Typically the analogue is immobilised (e.g. on particles or beads). The analogue is selected to have a lower affinity than the ligand for the anti-ligand monoclonal antibody. The assay thus works on the principle of immuno-competition, the presence of free ligand in the sample serving to decrease the amount of labelled antibody which becomes associated with the ligand analogue.
WO 91/05262 discloses a device and method for detecting the presence of molecular analytes in a fluid (especially e.g. steroids, and other low molecular weight analytes). Typically, aqueous biological samples are drawn along a test strip by capillary action. As the sample advances, it carries a labelled analyte from an area of storage at one end of the strip to a first binding means, which is an anti-analyte antibody. In the absence of free analyte in the sample, the labelled analyte (e.g. analyte/enzyme conjugate) will remain bound to the first binding means. However, if free analyte is present in the sample it will tend to displace the labelled analyte (or at least, compete therewith for binding sites on the first binding means) such that some labelled analyte will be bound to the second binding means, which is an anti-enzyme antibody. Colour is developed by placing the strip in an appropriate substrate solution.
EP 0,383,313 discloses a composition and assay method "for measuring haptens, antigens or antibodies by means of a competitive binding method". The invention disclosed therein requires that either the antibody or its ligand is labelled.
However, useful as such assays are, the requirement for labelling is disadvantageous. Radio-labels represent obvious hazards in handling and disposal. Enzyme or other active labels may deteriorate during storage, affecting the sensitivity of the assay. Use of coloured particles causes problems in that the relatively large surface area of the particles introduces non-specific binding sites which can affect the accuracy of the assay.
The present invention seeks to reduce these difficulties by providing an assay method and device which do not require the use of conventionally-labelled reagents.